Preparation of platinum nanoparticles and effect on c. elegans lifetime.

Preparation and assembly of "cotton-like nanogold" fibers.

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

One of the greatest challenges in electron microscopy is combining reliable sample preparation with adequate penetration of nanoparticle probes into cells and tissues to label interior structures. Recent advances in rapid freezing and fixation by freeze substitution have allowed structural and cell biologists to apply these more reliable modes of sample preparation to many specimens and scientific problems. Meanwhile, progress in electron tomography means that cellular images can now be generated with resolution approaching 4 nm in 3D; however, the ability to localize macromolecules within these well-fixed, well-resolved samples is limited. Using light fixation and low temperature embedding with appropriate resins, immunogold methods can recognize antigens at the section surface: however, labeling is confined to the section surface, and does not extend throughout its depth. Small, electron-dense markers, like Nanogold®, can enter a living cell, and once there serve as reliable tracers for endocytic activity; however, they are usually too small to visualize in the context of a cell, and autometallographic enhancement methods are not applicable to these samples.

Morphew, Bjorkman and co-workers have developed a method for silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature, based on in vitro tests of reagents and conditions. This method was applied to an in vivo system: Nanogold® was used to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic. Their method and results are described in a recent paper in the Journal of Microscopy.

The components of the silver enhancement solution were prepared in separate tubes as saturated solutions of silver nitrate (0.04%), hydroquinone (0.3%), citric acid (0.4%) and sucrose (0.1%), all in pure acetone. Solutions were protected from light and mixed on a rocking platform for 4 hours, then cooled to 20°C overnight. Cooled solutions were centrifuged at 600 X g for 10 minutes to remove any insoluble residues, then cooled to 50°C. Appropriate volumes of the four components were mixed in pre-cooled containers.

The reagents were first evaluated using dilutions of Non-functional Nanogold solution applied to blotting paper which were enhanced and examined visually, and using similar dilutions applied to EM grids which were examined by electron microscopy; finally, the reagents were tested using Ptk1 cells, grown on Formvar-coated, carbon-stabilized EM grids. After they had reached approximately 70% confluence, these cells were detergent-extracted in 0.1% Triton-X100 in PIPES buffer, fixed with paraformaldehyde and glutaraldehyde, then incubated with a primary anti-tubulin antibody diluted 1:10, followed by Nanogold-IgG goat anti-mouse IgG diluted 1:20. Grids with labeled cells were dehydrated into acetone by progressively lowering the temperature while raising the concentration of the solvent to 100% acetone at 50°C, then soaked overnight at 50°C in the enhancement solution. Some were then rinsed with acetone at 50°C, and others allowed to warm to 30°C before rinsing. Grids were then dried by the critical point method and examined by transmission electron microscopy at 200 kV.

For In vivo enhancement, Nanogold particles, conjugated to the Fc moiety of IgG, were fed to newborn rats. 200300 µL of rat Fc Nanogold conjugates at approximately 2 µM in 20 mM sodium phosphate with 1.2 mM CaCl2, 0.5 mM MgCl2, 0.25 mM MgSO4, pH 6.0 at 37°C were fed to 12-day-old Sprague Dawley rats that had fasted 3 hours before feeding. After 120 minutes, the rats were anesthetized with CO2, sacrificed, and a small segment of the duodenum was excised, cut into pieces (2 mm), transferred to freezing planchettes and frozen in a high-pressure freezer. Hexadecene was used as a filler to occupy empty space within the planchette-specimen sandwich. Frozen planchettes were separated using a cooled no.11 scalpel blade and transferred under liquid nitrogen to vials containing a frozen solution of 2% glutaraldehyde and 0.01% tannic acid dissolved in acetone. Vials with samplewere transferred to a pre-cooled freeze substitution machine at -90°C for two days. Samples were warmed to 50°C over 6 hours and were rinsed three times with cold acetone.

The four components necessary for silver enhancement of small gold were cooled, and mixed by adding two parts of silver nitrate, two parts of hydroquinone, one part of citric acid and one part of sucrose. The mixture was then cooled to 50°C in the dark and added to each 1.5 mL sample vial. After 12 hours at 50°C, the samples were warmed to 30°C over 6 hours, rinsed 3 times in cold acetone, post-fixed with 0.5% glutaraldehyde containing 0.1% uranyl acetate, then warmed to room temperature over 612 hours and rinsed in acetone.

Samples were infiltrated with epoxy resin, and tissue pieces flat-embedded between two treated microscope slides. After polymerization of the resin, small pieces of embedded tissue were excised and remounted onto blank epoxy stubs. Ribbons of 300-nm sections were collected on Formvar-coated copper slot grids and post-stained with uranyl acetate and Reynolds lead citrate. Sections were imaged by transmission electron microscopy at 300 kV. Areas containing the intestinal lumen were imaged through serial tilts 60?, and tomographic reconstructions computed.

In the preliminary blotting experiments at 4°C, visible dark spots developed in one hour, but at lower temperatures (-20 or -50°C) longer development times of 8-12 hours were required. TEM examination of enhanced Nanogold on grids showed a size range of 4 to 13 nm for the enlarged particles, and similar enhancement was observed in the Ptk1 cells, showing penetration and enhancement even of interior structures. Longer times or higher temperatures produced larger particles and background autoucleation. In the in vivo experiments, similar penetration and enhancement was observed, although the particle size on average was smaller than that found in the in vitro experiments; the enlarged particles were between 3 and 8 nm in size. Sample preservation appears excellent, making this a potentially useful method for high-quality electron microscopic labeling.

People frequently ask us which of our silver enhancers they should use for their particular application, what the differences between them are - and when it is better to use gold enhancement. There is considerable overlap between their uses, but they are based on different formulations, and differ significantly in their properties.

HQ Silver is formulated for the most uniform development, with the least perturbation of specimen ultrastructure. It contains a protective colloid, in order to control the rate of diffusion of the reactive species; this ensures that particles are enlarged uniformly, so that the enlarged particles have the least possible variation in size. It also permits the use of a near-neutral pH; this gives fast development, ensuring that the highest proportion of particles is developed; and because this reagent also has low ionic strength, it provides excellent ultrastructural preservation. This makes it ideal for electron microscopy applications where labeling density and size uniformity are critical, especially quantitative labeling applications, and also for delicate specimens with low fixation or sensitivity towards pH or ionic strength. However, this reagent is sensitive to sunlight or direct light, and therefore should be used in a darkroom with a safelight, or in diffuse light; for example, with the blinds closed or curtains drawn enough to leave sufficient light to see what you are doing.

LI Silver is formulated to give the most complete and specific development with the greatest degree of particle enlargement: it is formulated with components designed to minimize non-specific silver deposition or interaction with other features within the specimen. It requires a longer time than HQ Silver to achieve full development, and it is light insensitive, so it may be used in a fully lighted laboratory. It is intended for the highest staining and detection sensitivity in optical applications, including light microscopy, immunoblotting and gel staining. Because it develops more slowly and is light insensitive, it is ideally suited to applications where monitoring is needed; you can view the progress of the silver enhancement either by eye or in the light microscope, then stop when staining reaches the desired point either by rinsing with water, or treatment with freshly prepared 1 % sodium thiosulfate solution.

The key features and differences between these to reagents are illustrated and summarized below:

But...we also offer gold enhancement, an alternative process in which gold, rather than silver, is deposited onto gold particles. The different properties and reactivity of gold, and its stability towards physiological ions such as halides and phosphates, means that gold enhancement has advantages for several applications. If you are planning to use the following techniques, you should consider using GoldEnhance

Procedures where osmium tetroxide treatment is required and etching of silver-enhanced gold by osmium has been a problem; unlike silver, gold is not etched by osmium.

Enhancement of gold-labeled specimens, such as cultured cells, on metal substrates. While silver can be deposited onto the metal substrates, gold usually is not.

Some light microscopic applications, particularly in situ hybridization (ISH). Gold enhancement, used to visualize gold labeling as an ISH detection method, frequently produces lower background than silver enhancement, resulting in a cleaner or more specific signal.

Both silver and gold enhancement also work well with other immunogold reagents, including colloidal gold particles from other manufacturers. However, please note that both methods require the presence of gold nanoparticles. You cannot use silver enhancement on its own as a replacement for the formaldehyde-based "silver staining" methods: the chemistry of the process is different. In the formaldehyde silver staining system, the silver is deposited directly onto the biological molecule in question, and all the molecules on the gel are stained and visualized. In silver enhancement, the catalyst and nucleating center for silver deposition is an immunogold particle, and therefore silver enhancement will visualize only molecules linked to gold nanoparticle labels.

Please let us know if you need advice or assistance on selecting the best reagent for your application. We will be glad to advise.

Continuing the theme of silver enhancement, Sousa, Leapman and co-workers followed up their recent study on 3-D Distribution of Nanogold® and Undecagold by STEM Tomography with a study, reported recently in the Journal of Structural Biology to investigate the quantitative distribution of heavy and light atoms in heavy atom stained specimens, indicating the extent to which such small heavy atom cluster labels may be visualized in the presence of metallic stains. It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins.

The methods for determining the relative numbers of light and heavy atoms were tested on cells of the smallest known eukaryote, Ostreococcus tauri.O. tauri is a plant cell of diameter 12 µm. Specimens were prepared by immersion in a cryopreservative solution consisting of 15% sucrose, then freezing the mixture in a high-pressure freezing machine. The frozen blocks were processed at low temperature in a Leica EM-AFS freeze-substitution system using a solution of acetone containing 1.0% osmium tetroxide. After freeze-substitution for 3 days, the specimens were gradually warmed to room temperature and embedded in EponAraldite by graded exchange of the acetone. Specimens were polymerized in 100% resin by heating to 60°C for two days, sectioned to a nominal thickness of 3040 nm with a Leica Ultracut E ultramicrotome, and mounted on 300 mesh grids. Some of the sections were stained with lead citrate for 5 minutes, followed by staining with uranyl acetate for another 5 minutes; after each staining step, the sections were washed nine times in three 50 ml volumes of de-ionized water.

STEM images were acquired with a transmission electron microscope (FEI, Hillsboro, OR, USA) operating at an acceleration voltage of 300 kV and equipped with a field-emission gun. This microscope is fitted with a Model 3000 in-column HAADF detector (Fischione, Export, PA, USA) situated after the projection-lens system
and above the viewing screen. The HAADF detector incorporates a single crystal yttrium aluminum perovskite scintillator optically coupled to a photomultiplier tube. Selected specimen regions from both unstained and stained osmium-fixed O. tauri were first pre-exposed to a broad electron beam in TEM mode in order to stabilize shrinkage of the plastic section. STEM images were then collected from these regions with an electron dose per pixel of 2.5 x 104 e/nm2. The image dimensions were 2048 X 2048 pixels with a pixel size 0.87 nm. The convergence semi-angle of the illumination was 12 mrad. For each selected specimen region two STEM images were acquired, one with an ADF detector inner semiangle of 16 mrad, and another with a detector inner semiangle of 36 mrad. The outer semi-angle of the ADF detector was five times the inner angle. Control over the collection angles was achieved by varying the magnification of the diffraction pattern (camera length).

The same specimen regions of O. tauri selected for 2D STEM imaging were also used to acquire STEM tomographic tilt series. 10 nm fiducial markers were deposited on both sides of the sections before image collection. Tilt series were acquired from -60° to +60° with steps of 2°, with a pixel size of 0.87 nm, with an integral electron dose per pixel of 4 X 104 e/nm2, and with a fixed ADF detector inner semi-angle of 16 mrad. Data were collected using FEI STEM tomography software. For 3D reconstruction, tilt series were first aligned with the IMOD computer program using gold fiducial markers and reconstructed by weighted back-projection. The reconstructed tomograms were used to determine the thickness of the selected specimen regions. Finally, an additional STEM tomographic tilt series was acquired from a stained osmium-fixed O. tauri cell. The parameters used to acquire the tomographic tilt series were identical to those above, except the pixel size of the images was 0.43 nm and the integral dose was 1.7 X 105 e/nm2. Alignment of the tilt series was done using fiducial markers in IMOD and tomographic reconstruction was performed using the simultaneous iterative reconstruction tomography (SIRT) algorithm used previously. After alignment, but before reconstruction, the fiducial markers were removed from each tilt image to eliminate ghost streaks in the tomogram. The SIRT-reconstructed tomogram was then used to estimate the distribution of heavy metal stain atoms in 3D.

Testing the method on a specimen of 20 nm carbon film upon which had been deposited 1.4 nm Nanogold, the method provided a value of 63 ± 13 for the number of gold atoms in a Nanogold particle - consistent with other observations on similar clusters. The authors then used their approach to quantitate osmium atoms in osmium stained specimens. In sectioned cells that have been stained only with osmium tetroxide, they found an average of only 1.2 ± 0.1 Os atom per nm3, corresponding to an atomic ratio of Os : C atoms of approximately 0.02. A this density of osmium, the small heavy atom clusters of Undecagold and Nanogold can be detected, and this indicates that STEM tomography is capable of visualizing these labels in lightly stained specimens.

The Saccharomyces cerevisiae protein Bro1p is required for sorting endocytic cargo to the lumen of multivesicular bodies (MVBs). Cellular components involved in MVB biogenesis support late events during retroviral budding, where they are recruited via conserved peptides within "Late Domains" of viral Gag proteins. The p6 Late Domain of HIV binds the ESCRT-I subunit TSG101 through a PTAP peptide and binds the Bro1p-related protein Alix via a YPD-LXXLF motif. This indirectly suggests that Alix might also contribute to MVB sorting. However, although Alix is structurally related to Bro1, localizes to endosomes, and binds ESCRT proteins in vitro, up to now functional studies have failed to identify a role for Alix in MVB formation. To investigate whether Alix or similar proteins participate in endosomal sorting, the authors attached a retroviral peptide to bind Alix to a reporter receptor. HIV p6 was attached to the cytoplasmic domain of transferrin receptor (TfR), which normally cycles between the endosome and the cell surface

Although TfR-GFP distribution was similar to endogenous TfR, most p6-TfRGFP was found to localize to intracellular foci, consistent with relocation to later endocytic compartments. Electron microscopy was used to determine whether HIV p6 supports inward vesiculation of cargo within these compartments. For Immunogold labeling, the cells were fixed with 4% PFA in 0.1M HEPES, blocked with PBS containing 1% BSA, 0.15 glycine and 0.2% (wt/vol) saponin, then labeled with primary antibodies, followed by secondary Nanogold-Fab' fragments. The specimens were then enhanced with GoldEnhance EM for 57 minutes: this generated gold particles with a range of sizes. Samples were then postfixed with reduced osmium tetroxide, processed for standard epon embedding and examined by using a transmission electron microscope (FEI) at 100 kV. For analyzing p6-TfR-containing compartments, mature MVB/late endosomes were classified as vacuoles containing internal membrane content but lacking the extensive membrane lamellae characteristic of lysosomes.

Most p6-TfR-GFP localized to the lumen of vacuoles. Myc-p6-TfR, epitope tagged on the cytoplasmic domain, also labeled the lumen, consistent with the chimera sorting to this location. The morphology of these vacuoles, with abundant internal vesicles and some additional content, was similar to that of mature MVBs/late endosomes in untransfected cells. The chimera was sorted efficiently away from the early endosome to the lumen of late endocytic compartments. This sorting was not prevented by depleting Alix, but instead required the Alix-related protein His domain phosphotyrosine phosphatase (HD-PTP)/His-Domain/Type N23 protein tyrosine phosphatase (PTPN23). Depletion of HD-PTP also reduced transfer of fluid-phase markers and EGF receptor to lysosomes, caused the accumulation of ubiquitinated proteins on endosomal compartments and disrupted the morphogenesis of MVBs. Rescue experiments using an RNAi-resistant version of HD-PTP and HD-PTP mutants demonstrated an essential role for the HD-PTP Bro1 domain, with ESCRT-III binding correlating with full biological activity. The aim of this study was to establish whether mammalian proteins related to S. cerevisiae Bro1p are involved in MVB sorting. The most likely candidate was Alix, based on its pattern of molecular interactions and ability to support virus budding. However, although attachment of a peptide capable of binding Alix diverts TfR to the MVB pathway, sorting of this chimera does not seem to depend strictly on Alix. Depletion of Alix also only moderately affected EGF trafficking; but transport of both cargoes was profoundly affected upon knockdown of HD-PTP. It could not be determined whether HD-PTP supports p6-TfR trafficking directly, because binding could not be detected between HD-PTP and p6, although HD-PTP may bind p6 with too low an affinity for detect in this experiment. These findings suggest that HD-PTP is a key regulator of endocytic trafficking in which ESCRT-III binding is important, but not strictly essential.

Nanogold with silver enhancement can be used in conjunction with enzymatic DAB staining as a double labeling method, and Endo and co-workers report more results using this approach in their recent paper to localize proteins involved with dendritic Ih, a protein which ensures high-fidelity dendritic spike responses of motion-sensitive neurons in rat superior colliculus. Coronal rat brain sections (50 µm thick) were cut into 25 mM PBS with a vibrating microslicer; Fluorescence signals were observed in the sSC region by a fluorescence microscope, and GFP-expressing WFV cells were identified based on their morphology and photographed. After fluorescence observation, sections were washed three times in 0.1 M phosphate buffer and cryoprotected in 25% sucrose and 20% glycerol in 0.02 M PB for 3 hours. After freeze-thawing in liquid nitrogen, sections were washed in 50 mM TBS three times, and incubated for 1 hour at room temperature in TBS containing 20% normal goat serum to block nonspecific binding. After blocking, the sections were incubated with guinea pig anti-HCN1 antibody (1 µg/ml) (Notomi and Shigemoto 2004) and rabbit anti-GFP antibody (0.05 µg/ml) in TBS containing 1% normal goat serum for 24 h at 4°C. After washing in TBS, sections were incubated with Nanogold-labeled anti-guinea pig IgG and biotinylated anti-rabbit IgG (Vector, Burlingame, CA) secondary antibodies at 4°C overnight, then treated with HQ Silver. Finally, the sections were incubated with ABC-Elite (Vector) and then 0.02% diaminobenzidine-4HCl solution in 0.003% H2O2. After osmication, the immunostained sections were block-stained with uranyl acetate, dehydrated, and flat-embedded in Epon (Durcupan, Fluka). Ultrathin sections containing GFP-expressing WFV cells were prepared and examined for HCN1 signals with a transmission electron microscope. Blocking Ih suppressed the initiation of short- and fixed-latency dendritic spike responses, and led instead to long- and fluctuating latency somatic spike responses to optic fiber stimulations. These results suggest that the dendritic Ih facilitates the dendritic initiation and/or propagation of action potentials and ensures that WFV cells generate spike responses to distal synaptic inputs in a sensitive and robustly time-locked manner, probably by acting as continuous depolarizing drive and fixing dendritic membrane potentials close to the spike threshold. These functions are different from known functions of dendritic Ih found in hippocampal and neocortical pyramidal cells, where they spatiotemporally limit the propagations of synaptic inputs along the apical dendrites by reducing dendritic membrane resistance. These results indicate new functional aspects of Ih, and these dendritic properties are likely critical for visual motion processing in these neurons.

Could metal nanoparticles be the elixir of life? In a recent Mechanisms of Ageing and Development, Kim and group report that platinum nanoparticles (nano-Pt) can significantly extend the lifetime of c. elegans. Platinum particles are a superoxide dismutase (SOD)/catalase mimetic, and various data have shown extension of the Caenorhabditis elegans lifespan by antioxidant treatment. The present study was designed to elucidate the survival benefit conferred by nano-Pt, as compared to the well-known SOD/catalase mimetic EUK-8. At 0.5 mM, nano-Pt significantly extended the lifespan of wild-type N2 nematodes and at 0.25 and 0.5 mM, nano-Pt recovered the shortened lifespan of the mev-1(kn1)mutant, which is due to excessive oxidative stress. In both instances, EUK-8 at 0.05, 0.5, and 5 mM did not extend nematode lifespan. Even when 0.4 M paraquat was loaded exogenously, nano-Pt (0.1 and 0.5 mM) and EUK-8 (0.5 and 5 mM) were effective in rescuing worms. Moreover, 0.5 mM nano-Pt significantly reduced the accumulation of lipofuscin and ROS induced by paraquat. The authors measured the in vitro dose-dependent quenching of O2 and H2O2, indicating that nano-Pt is a more potent SOD/catalase mimetic than EUK-8. Nano-Pt prolonged the worm lifespan, regardless of thermotolerance or dietary restriction. Taken together, this shows that nano-Pt has interesting anti-ageing properties.

Meanwhile, Fu and colleagues have reported the synthesis of 'cotton-like' gold fibers and some useful properties of these constructs. A simple approach for the large-scale synthesis of cotton-like 'nanogold' was developed using liquid-based process at room temperature is described. The gold fibres have diameters in the range of 1540 nm and lengths longer than 50 ?m, entwined into a complex mass. The first step of the preparation procedure involves the formation of gold seeds by reducing HAuCl4 with NaBH4 in a solution containing an appropriate quantity of cetyltrimetylammonium bromide (CTAB). Next, the gold seeds continuously grow to form cotton-like nanogold in a solution containing HAuCl4, CTAB and L-ascorbic acid (AA) under an ultrasonic radiation field. The cotton-like nanogold has observable fluorescence, which might have great significance in biological and medical applications. HRTEM images showed that many defects and twin crystals exist in the turning junctions of as-prepared cotton fibres, which might be the reason the turning junctions in the fibres became stable.